Crotoxin B: Heterologous Expression, Protein Folding, Immunogenic Properties, and Irregular Presence in Crotalid Venoms

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.


Introduction
Crotoxin is a PLA 2 isolated and purified originally from the venom of the snake Crotalus durissus terrificus. It is generally found as a heterodimeric complex consisting of two subunits: the subunit CA, also called Crotapotin, and the subunit CB, also called Crotoxin B. The two subunits (CA/CB) are linked by non-covalent bonds [1,2]. The subunit CA is not toxic, but the subunit CB generally has PLA 2 activity and is toxic to mammals. The lethality of crotoxin is favored and increased with the formation of the complex acids at its N-terminus (MRGSHHHHHHGSENLYFQG). The mature peptide contained the restriction site BamHI (GS), a His-Tag region added by the pQE-30 vector, and the proteolytic TVE site (ENLYFQG), resulting in 141 residues of the His-Tagged neurotoxin ( Figure S1). The construction was named HisrCrotoxin B, and it retains the conserved residues His48, Asp49, Tyr52, and Asp99 of the common PLA 2 active site (here, His47, Asp48, Tyr51, and Asp89), which were agreed to be characteristic of type II phospholipases [19] (Table 1). Table 1. Amino acid sequence of HisrCrotoxin B compared to the native Crotoxin B.

Expression, Purification, and Protein Folding of HisrCrotoxin B
The heterologous expression of HisrCrotoxin B was achieved using the E. coli Origami strain. HisrCrotoxin B was found mainly in inclusion bodies, and it was recovered after using agarose nickel affinity. HisrCrotoxin B was reduced with 1,4-dithiothreitol (DTT), and it was purified using a high-pressure liquid chromatography (HPLC) system using reverse-phase chromatography (RP-HPLC). The protein fraction with a retention time of 48 min was collected ( Figure 1). An analysis by 15% SDS-PAGE showed that the protein fraction had a similar apparent molecular mass to the expected molecular mass ( Figure 2). Furthermore, the protein fraction showed an experimental molecular mass of 16,421.7 Da, obtained by mass spectrometry. It corresponded to the expected molecular mass for the 6His-tagged HisrCrotoxin B in its reduced form. The protein yield of HisrCrotoxin B was 1 mg/L. After purification by HPLC, the protein was folded in vitro Time (min) Absorbance (230/280 nm)  (2) reduced HisrCrotoxin B protein; (3) folded recombinant protein; (4) native Crotoxin B protein of C. tzabcan. A total of 10 µg of each sample reduced with β-mercaptoethanol was used.

Evidence of Similar Structure between Native Crotoxin B and HisrCrotoxin B
It is important to note that a heterologously expressed protein containing 14 cysteines could theoretically form up to 135,135 isoforms if considering only the disulfide pairing possibility. The number of possible Cys-Cys structural forms for a protein rich in cysteines increases with the number of cysteines in the molecule; consequently, to obtain a structural enduring and in vivo functional recombinant protein, the right structure must be preserved. The oxidized HisrCrotoxin B and the native Crotoxin B (nCrotoxin B) were enzymatically digested, first with trypsin and then with Glu-C, to compare their structures. The digested protein fractions were separated by RP-HPLC, and the most prominent peptide fractions were analyzed by mass spectrometry and Edman degradation. The HPLC elution profiles displayed digested fraction patterns with similar retention times having the same molecular masses, yielding the same amino acid sequences for both the native Crotoxin B and the HisrCrotoxin B ( Figure 3). If different HPLC elution profiles were displayed for any of the two proteins, it could indicate a different disulfide pairing arrangement. Furthermore, the protein fraction showed an experimental molecular mass of 16,421.7 Da, obtained by mass spectrometry. It corresponded to the expected molecular mass for the 6His-tagged HisrCrotoxin B in its reduced form. The protein yield of HisrCrotoxin B was 1 mg/L. After purification by HPLC, the protein was folded in vitro following the conditions used by Fonseca et al. [20]; that is, the folding reaction contained 12 µM HisrCrotoxin B, 0.05 mM Tris-base, 2 M GndHCl, 1 mM CaCl 2 , and 1.7:0.2 mM Cys:Cys-Cys as a par redox, pH 6.0. The solution was continuously stirred for 48 h at room temperature. After that, the solution was centrifugated for 10 min at 16,000× g in an Eppendorf Centrifuge 5418 and purified again by RP-HPLC using the abovementioned same conditions. HisrCrotoxin B, in its oxidized form, eluted at a retention time of 44 min ( Figure 1).

Evidence of Similar Structure between Native Crotoxin B and HisrCrotoxin B
It is important to note that a heterologously expressed protein containing 14 cysteines could theoretically form up to 135,135 isoforms if considering only the disulfide pairing possibility. The number of possible Cys-Cys structural forms for a protein rich in cysteines increases with the number of cysteines in the molecule; consequently, to obtain a structural enduring and in vivo functional recombinant protein, the right structure must be preserved. The oxidized HisrCrotoxin B and the native Crotoxin B (nCrotoxin B) were enzymatically digested, first with trypsin and then with Glu-C, to compare their structures. The digested protein fractions were separated by RP-HPLC, and the most prominent peptide fractions were analyzed by mass spectrometry and Edman degradation. The HPLC elution profiles displayed digested fraction patterns with similar retention times having the same molecular masses, yielding the same amino acid sequences for both the native Crotoxin B and the HisrCrotoxin B (Figure 3). If different HPLC elution profiles were displayed for any of the two proteins, it could indicate a different disulfide pairing arrangement.   Furthermore, to compare the secondary structure of HisrCrotoxin B, the native Crotoxin B from C. tzabcan and a native PLA 2 that was isolated from Bothrops ammodytoides venom were analyzed by circular dichroism (CD). The subunits, HisrCrotoxin B and the native Crotoxin B, showed high absorption for β-structures and relatively low content for α-helix secondary structure ( Figure 4). According to a CD deconvolution program (BeStSel, https://bestsel.elte.hu, (accessed on 5 May 2022), the secondary structure contents were 13.6, 37.0, 18.5, and 30.9; 9.5, 28.3, 15.7, and 45.4; and 19.7, 22.7, 15.7, and 41.9 of α-helix, β-structures, β-turns, and other structures, respectively, for HisrCrotoxin B, native Crotoxin B, and nPLA 2 , respectively. So far, all pit viper venom PLA 2 s were found to contain a greater proportion of α-helix, characterized by a positive band at 198 nm and negative ellipticities at 208-222 nm [21].
(Vydac 214 TP 4.6 × 250 mm, Columbia, MD, USA) using 0.1% trifluoroacetic acid (TFA) in water as solvent A, and 0.1% TFA in acetonitrile as solvent B. The gradient was run from 20 to 60% solvent B for 40 min at 1 mL/min, and the peptide fragments were detected at 230 nm. The HPLC fractions collected were analyzed using mass spectrometry and Edman degradation. (C) The amino acid sequence of HisrCrotoxin B shows in bold the amino acid sequences detected by mass spectrometry and Edman degradation in the digested fractions.
In 1989, Aird et al. [22] revealed comparative spectroscopic studies of four crotoxin homologs and their subunits. They examined the secondary structures of four native crotoxins and their purified subunits from the venoms of C. durissus terrificus, C. vegrandis, C. s. scutulatus, and C. viridis concolor by circular dichroism. The obtained CD spectra of the isolated subunits of Crotoxin B were decreased slightly in α-helix, while they were increased in β-sheet structures, relative to intact native crotoxins [22]. For example, the native subunit Crotoxin B from C. durissus terrificus had 16, 51, and 33% of α-helix, β-sheet The native PLA 2 was isolated from Bothrops ammodytoides venom [15,16].
In 1989, Aird et al. [22] revealed comparative spectroscopic studies of four crotoxin homologs and their subunits. They examined the secondary structures of four native crotoxins and their purified subunits from the venoms of C. durissus terrificus, C. vegrandis, C. s. scutulatus, and C. viridis concolor by circular dichroism. The obtained CD spectra of the isolated subunits of Crotoxin B were decreased slightly in α-helix, while they were increased in β-sheet structures, relative to intact native crotoxins [22]. For example, the native subunit Crotoxin B from C. durissus terrificus had 16, 51, and 33% of α-helix, β-sheet structure, and other structures, respectively. In this work, according to the CD deconvolution program used (BeStSel), the secondary structure contents of the native Crotoxin B were 9.5, 28.3, 15.7, and 45.4 of α-helix, β-structures, β-turns, and other structures, respectively. The secondary structures observed for all four native Crotoxin B by Aird et al. [22] are quite similar to the native Crotoxin B from C. tzabcan and the recombinant HisrCrotoxin B. Contrary to the CD of the subunits of Crotoxin B (native and recombinant), the intact native Crotoxin from C. durissus terrificus had 29, 23, and 48% of α-helix, β-sheet structure, and other structures [22], respectively, which had higher α-helix and lower content of β-sheet structure values like the control used (PLA 2 from the viperid B. ammodytoides). Comparing the low content of α-helix of HisrCrotoxin B and the native Crotoxin B obtained here by CD with that of the α-helix obtained by X-ray crystallography of native Crotoxin B [2,[23][24][25] and other PLA 2 s, they may differ in the fact that the X-ray crystallography structures are practically obtained in a semi-solid/solid state, maximizing the hydrogen bonds of the secondary structures. On the other hand, the secondary structure obtained by CD is obtained in a more dynamic liquid environment; however, the CD deconvolution algorithms are based on few minima of wavelength light absorption, such as 208 and 222 nm, and in the number of other proteins in the CD database used. Therefore, the content of α-helix secondary structure obtained by CD or X-ray crystallography may have some deviations. To have a better comparison, in this work, we compared the CD of both HisrCrotoxin B and the native Crotoxin B, and also with a relate native PLA 2 , which has a slightly higher content of α-helix, but still lower than that of other viperid PLA 2 s such as that of BmajPLA2-II, a basic Lys49 PLA 2 homologue [26].

Rabbit Immunization, Antibody Recognition, Antibody Titers, and Inhibition of Phospholipase Activity
The rabbits were immunized each with 7.3 mg of HisrCrotoxin B. After 3 months of immunization, the rabbits were bled, and the serum was used to observe its capacity to recognize HisrCrotoxin B and native crotoxins from Crotalus spp. venoms. Figure 5A shows the protein composition of venoms from several species of Crotalus. Native crotoxins, according to their observed apparent molecular masses, might have from none to one-third of their respective venom composition (see red square marks in Figure 5B). The apparent molecular mass of HisrCrotoxin B ( Figure 5B, lane 2) looked larger than the other natural crotoxins. This may have been caused by the extra basic residues (6His-tag) at the N-terminal of HisrCrotoxin B, which might retard the protein migration, as observed previously [27]. Nevertheless, the mass spectrometry confirmed the expected molecular mass for HisrCrotoxin B. Yet, Figure 5B shows the recognition of the rabbit anti-HisrCrotoxin B to venom proteins from Crotalus species (C. tzabcan and C. mictlantecuhtli), including native Crotoxin B. However, it did not recognize venom proteins from C. culminatus, Bothrops asper, Micrurus laticollaris (elapid), and Chihuahuanus crassimanus (scorpion). The antibody recognition was observed in proteins from 10 to 15 kDa, representing the native crotoxins from Crotalus spp. venoms, but C. culminatus did not contain Crotoxin B [28]. The recognition of Crotoxin B within the viperid venoms by anti-HisrCrotoxin B was evaluated in ELISA plates to support the previous observations ( Figure 6A). The IC50 values represented the serum concentration (µg/mL) required to obtain half of the colorimetric response. They had implicit the concentration of an inhibitor where the response (or binding) was reduced by half ( Table 2). It is important to note that ELISA experiments The recognition of Crotoxin B within the viperid venoms by anti-HisrCrotoxin B was evaluated in ELISA plates to support the previous observations ( Figure 6A). The IC 50 values represented the serum concentration (µg/mL) required to obtain half of the colorimetric response. They had implicit the concentration of an inhibitor where the response (or binding) was reduced by half ( Table 2). It is important to note that ELISA experiments allow the recognition of structural epitopes. In other words, the titers represent the needed concentration of antibodies (IC 50 in µg/mL) to recognize structural similarities in Crotoxin B within the viperid venoms. Table 2 summarizes the IC 50 values. The venoms C. tigris, C. s. salvini, and C. mictlantecuhtli had the best IC 50 values (lower µg/mL). M. melanurum, C. s. scutulatus, and C. basiliscus had significant recognition of Crotoxin B, but C. atrox, C. tzabcan, O. smaragdinus, and C. m. nigrescens had poor recognition (higher µg/mL) of Crotoxin B. Moreover, the anti-HisrCrotoxin B antibodies were tested against the phospholipase activity of C. tzabcan. Therefore, different concentrations of anti-HisrCrotoxin B were preincubated for one hour at 37 °C with 15 µg of the native protein. Once such a time had elapsed, the enzymatic activity assay was carried out by a conventional titrimetric assay. As mentioned previously, the phospholipase activity of the native Crotoxin B was determined at 9.8 ± 2.5 U/mg. In comparison, the activity of the same pre-incubated protein plus 800 µg of anti-HisrCrotoxin B antibodies was 2.3 ± 0.5 U/mg, representing only 23% of original PLA2 activity. In the same way, the enzymatic activity was performed with the complete venom of C. tzabcan, which had an activity of 9 ± 1 U/mg, while the PLA2 activity of the whole venom pre-incubated plus 800 µg of anti-HisrCrotoxin B antibodies was 2.9 ± 0.9 U/mg, representing 32% of the original phospholipase activity of C. tzabcan venom ( Figure 6B).   Moreover, the anti-HisrCrotoxin B antibodies were tested against the phospholipase activity of C. tzabcan. Therefore, different concentrations of anti-HisrCrotoxin B were preincubated for one hour at 37 • C with 15 µg of the native protein. Once such a time had elapsed, the enzymatic activity assay was carried out by a conventional titrimetric assay. As mentioned previously, the phospholipase activity of the native Crotoxin B was determined at 9.8 ± 2.5 U/mg. In comparison, the activity of the same pre-incubated protein plus 800 µg of anti-HisrCrotoxin B antibodies was 2.3 ± 0.5 U/mg, representing only 23% of original PLA 2 activity. In the same way, the enzymatic activity was performed with the complete venom of C. tzabcan, which had an activity of 9 ± 1 U/mg, while the PLA 2 activity of the whole venom pre-incubated plus 800 µg of anti-HisrCrotoxin B antibodies was 2.9 ± 0.9 U/mg, representing 32% of the original phospholipase activity of C. tzabcan venom ( Figure 6B).

Neutralization Activity
Although the antibody recognition, by both Western blot and ELISA plates, and inhibition of phospholipase activity were evident, in the titimetric assay, towards Crotoxin B in crotalid venoms, the neutralization assays are needed to assess the relevance of HisrCrotox-inB as an immunogen. For that, an in vivo neutralization test was performed using mice (CD1, 18-20 g). Three median lethal doses (LD 50 ) of native Crotoxin B and crotalid venoms were challenged against different concentrations of anti-HisrCrotoxin B pre-incubated at 37 • C for an hour in a final volume of 200 µL with PBS, which were administered intravenously to the mice. Their survival was evaluated at 48 h after administration. 3LD 50 assessed with pre-immune antibodies or without anti-HisrCrotoxin B were used as a positive control, also incubated for an hour at 37 • C. Similarly, anti-HisrCrotoxin B against the complete venoms of C. tzabcan, C. s. salvini, and C. miclanthecuhtli were challenged using the same protocol. Table 3 shows the neutralizing potency, expressed as mean effective dose (ED 50 ) against native Crotoxin B, which was 1.5 mg/3LD50, while the ED 50 s against the complete venoms of C. tzabcan, C. s. salvini, and C. miclanthecuhtli were 1.5, 14, and 2.1 mg/3LD50, respectively. Thus, the venom neutralization was effective for venoms of C. tzabcan, C. s. salvini, and C. miclanthecuhtli. Although ELISA assays are far from being able to be used to predict antivenom neutralization, interesting data can be inferred from them. For example, the antibody recognition experiments were correlated with the neutralization assays. That is, the venoms of C. tzabcan, C. s. salvini, and C. mictlantecuhtli were recognized and neutralized (Table 3).
These data proved that HisrCrotoxin B could be a suitable immunogen to raise anti-Crotoxin B and anti-PLA 2 antibodies to decrease PLA 2 activity of C. tzabcan phospholipases, related to several toxic activities such as myotoxicity or interfering with the platelet function, at least in this C. tzabcan venom.

Irregular Presence of Crotoxin B in Crotalid Venoms
Although Crotoxin B is a characteristic component of the venom of crotalids, it was noticed in Western blots and the ELISA assays that some crotalid individual venoms such as C. atrox and C. m. nigrescens did not seem to contain Crotoxin B. To determine the absence of Crotoxin B in some crotalid species, SDS-PAGE and Western blots were performed ( Figures S2-S10). Moreover, a milligram of some crotalid pools of venoms was run by HPLC under the same conditions to compare retention times related to Crotoxin B from the venom of C. tzabcan. Figure 7 shows the RP-HPLC chromatograms of the whole venoms of selected viperid species C. tzabcan, C. s. scutulatus, Ophryacus smaragdinus, C. atrox, and C. m. nigrescens.
The venom from C. scutulatus scutulatus (52.8 min) contained a fraction with retention time and molecular mass (14,214.4 Da) like that of the venom from C. tzabcan (52.1 min), from which Crotoxin B was isolated. However, the venoms from Ophryacus smaragdinus, C. atrox, and C. m. nigrescens did not contain relevant fractions within such retention times. The molecular mass of 13,776.8 Da found for a fraction from C. atrox venom seemed to be a related PLA 2 with domains perhaps different from that of Crotoxin B. The venoms of O. smaragdinus and C. m. nigrescens contained fractions with retention times later than that of the 52.1-52.8 min; however, no molecular masses were found for such fractions. Variation in the venom composition over a species' geographic distribution is an integral part of intraspecific variation [28][29][30]. That is, venom composition variation in biological and biochemical activities among adults, as well as ontogenetic changes from juvenile to adults, has been observed. For example, juvenile venoms are more lethal and had higher percentages of crotamine and crotoxin than adults [31]. Moreover, it is known if diet, temperature, oxygen levels, humidity, and light cycles could affect such venom variation. Therefore, geographical differences may affect clinically because envenomations could present different symptomatology depending on the region. It is also important to know whether there is a geographic variation in the venoms used as immunogens because this might affect the antivenom quality [32]. Consequently, this work provides information to improve the quality of immunogens.

Conclusions
This work presents for the first time the use of a recombinant Crotoxin B subunit, Hisr-Crotoxin B, to produce rabbit serum antibodies against native crotoxins from Crotalus spp. venoms. The inhibition of PLA 2 activity, the circular dichroism spectrum of HisrCrotoxin B, and the neutralization of Crotalus spp. venoms suggest that the recombinant Crotoxin B keeps basic secondary structure domains, such as native Crotoxin B, making it possible to raise neutralizing antibodies. Therefore, HisrCrotoxin B could be used as an immunogen to raise antibodies against related C. tzabcan species. The use of recombinant toxins as immunogens to raise neutralizing antibodies against simple neurotoxic venoms such scorpions (Centruroides, Tityus, Androctonus) and spiders (Loxoceles) has already been reported [33,34]. In the same way, other recombinant proteins could be used to generate neutralizing antibodies to reduce the toxic activities caused by the envenomation of poisonous animals. Furthermore, the reactivity of the anti-HisrCrotoxin B on other Crotalus venoms provides an interest in the study on the possible neutralization of toxic activities related to this toxin. Although currently anti-snake venom production for therapeutics are polyclonal antibodies produced in animals mostly using the whole venom as the immunogen, new methodologies are under study [35]. The use of recombinant proteins to generate polyclonal and monoclonal antibodies or neutralizing fragments through molecular biology methods could be the future for antivenoms production [36]. Moreover, snake venom PLA 2 s such as Crotoxin B can be used as an experimental model for the development of anti-inflammatory drugs for therapy in humans [37]. Therefore, this report provides a proof of principle for taking advantage of recombinant immunogens for developing commercial antivenoms or drug leads.

Venom and Venom Gland
Venoms were obtained from the Instituto de Biotecnología and from the Herpetario Kiinam (register number DF-REP-208-10-08, Cd. de Mexico, Mexico). An adult specimen of Crotalus tzabcan was kept in good health conditions and in plastic cages at 27 • C of constant temperature. The snake was fed fortnightly with a mouse, and tap water was provided ad libitum. The light-dark cycles were 12 h. The venom collected by manual extraction was immediately vacuum dried and stored at −20 • C until use. A healthy specimen was selected to obtain one of the two venom glands and removed by surgical extraction after being anesthetized with ketamine-xylazine. Immediately after extraction of the venom gland, it was treated with RNAlater ® (Thermofisher, Asheville, NC, USA) and stored at −20 • C until use. After surgical intervention, the specimen recovered itself and remained healthy.

Bacterial Strains, Enzymes, and Plasmids
The XL1-Blue Escherichia coli strain was used for DNA cloning and plasmid propagation. The Origami E. coli strain was employed to express the recombinant PLA 2 . Plasmids pCR ® 2.1-TOPO ® (Invitrogen, Carlsbad, CA, USA) and pQE30 (Qiagen, Valencia, CA, USA) were used for cloning the Crotoxin B gene and production of the 6His-tagged recombinant HisrCrotoxin B, respectively. Restriction enzymes, Taq polymerase, and T4 DNA ligase were purchased from New England Biolabs (New England Biolabs, Ipswich, MA, USA).

RNA Extraction and Gene Assembly
The total RNA was extracted from a single venom gland of Crotalus tzabcan using the "Total RNA Isolation System" (Qiagen, Valencia, CA, USA). Based on the N-terminal sequence of the previously reported PLA 2 , specific oligonucleotides were designed to amplify the corresponding transcript [35]. The oligonucleotides were named Oligo Crotoxin B-dir (GAG GGG CAC CTG CTG CAA TTC, Tm 61.4 • C) and Oligo Crotoxin B-rev (GAC TTA GCA TGT CTC TGA AGG CCC, Tm 59.3 • C).
The obtained insert amplified by Taq polymerase was purified using the High Pure PCR Product Purification Kit (Roche, Basel, Switzerland), then ligated to the plasmid pCR ® 2.1-TOPO ® (Invitrogen, Carlsbad, CA, USA), and finally used to transform quimiocompetent XL1-Blue E. coli cells. Positive clones were selected on the basis of the size of the amplified segment by colony PCR (M13-Forwar and M13-Reverse oligonucleotides). After plasmid purification (High Pure Plasmid Isolation Kit, Roche, Basel, Switzerland), the integrity of the gene construction was verified by DNA sequencing.

Plasmid Construction for Expression
The gene construction to express the HisrCrotoxin B included recognition sequences for restriction enzymes used for cloning (BamHI and PstI) and a sequence encoding the TEV cleavage site. The designed transcript was subcloned into the pQE30 expression vector through the BamHI and PstI sites. The pQE30 vector introduces a polyhistidine-tag (6His) to facilitate product purification by affinity chromatography. The tobacco etch virus (TEV) cleavage sequence was conveniently placed between the 6His and the mature toxin to allow the cleavage of the whole recombinant toxin if necessary. The new pQE30-derived constructs were verified by sequencing from both sides. Quimiocompetent E. coli Origami cells were transformed with the corresponding plasmids by incubation for 30 min on ice, heat-shocked for 1 min at 42 • C followed by 5 min in ice, recovered for 1 h at 37 • C in SOC medium, and plated in LB containing 100 µg/mL of ampicillin. The constructions were named pQE30:HisrCrotoxin B, and their expression product was here abbreviated as HisrCrotoxin B.

Molecular Mass Determination of HisrCrotoxin B, Native Crotoxin B, and Peptides from the Enzymatic Digestions
The molecular mass identities of HisrCrotoxin B, Crotoxin B from C. tzabcan and other native Crotoxin B, and the enzymatically digested peptides were confirmed by mass spectrometry analysis. The protein fractions were reconstituted to a final concentration of 500 pmol/5 µL of 50% acetonitrile with 1% acetic acid and directly applied to a Thermo Scientific LCQ Fleet ion trap mass spectrometer (San Jose, CA, USA) using a Surveyor MS syringe pump delivery system. The eluate at 10 µL/min was split out to introduce only 5% of the sample into the nanospray source (0.5 µL/min). The spray voltage was set from 1.5 kV, and the capillary temperature was set to 150 • C. The fragmentation source was operated at 25-35 V of collision energy and 35-45% (arbitrary units) of the normalized collision energy, and the scan with a wide band was activated. All spectra were obtained in the positive-ion mode. The data acquisition and deconvolution were performed on the Xcalibur Windows NT PC data system (Thermo Fisher Scientific, Waltham, MA USA).

Enzymatic Digestions of HisrCrotoxin B and Native Crotoxin B
Either Crotoxin B from C. tzabcan or recombinant HisrCrotoxin B were separately digested and incubated with trypsin (4 µg/mL at 37 • C) for 16 h. Afterward, Glu-C was added (2.5 µg/mL at 37 • C) for 6 h more. The digested peptides were fractionated using an analytic C 18 reverse-phase column (Vydac 214 TP 4.6 × 250 mm, Columbia, MD, USA) running from solvent A (0.1% trifluoroacetic acid, TFA, in water) to solvent B (0.1% TFA in acetonitrile). The HPLC system previously described was used for this separation, and the gradient was run from 0 to 60% solvent B for 60 min at 1 mL/min; the proteins were detected at 230 nm. Additionally to the mass spectrometry analysis of the digested peptides (Section 4.6), N-terminal Edman degradation was performed on a Shimadzu PPSQ-31A (Shimadzu, Kyoto, Japan) automated gas-phase sequencer. A sample (60 µg) was dissolved in 10 mL of 37% CH3CN (v/v) solution and applied to TFA-treated glass fiber membranes, pre-cycled with Polybrene (Sigma-Aldrich Co. St. Louis, MO, USA).

Circular Dichroism
The secondary structure contents of a native PLA 2 and recombinant HisrCrotoxin B were calculated using circular dichroism spectroscopy (CD). Spectra were recorded at room temperature in quartz cells (1 mm-path) and wavelengths from 190 to 260 nm using a spectropolarimeter Jasco J-710 (Jasco, Japan). Data were collected every 1 nm at 50 nm/min. Each protein was dissolved in 60% trifluoroethanol up to a 0.6 mg/mL concentration. Trifluoroethanol improves secondary structure. The CD values correspond to the mean of three CD recordings. Finally, percentages of secondary structure were analyzed using the algorithms hosted online at BeStSel (Beta Structure Selection. webserver http://bestsel.elte.hu/index.php (accessed on 5 May 2022) [38].

Electrophoretic Analysis and Western Blotting
For electrophoretic analysis, SDS-PAGE under reducing conditions (4/15%) was performed according to the method proposed by Laemmli [39]. The protein staining was made with Coomassie Brilliant Blue. For Western blotting analysis, venom samples were first separated by SDS-PAGE (15%) and then transferred to a polyvinylidene difluoride membrane using a transfer apparatus Owl semi-dry system, 1 h at 400 mA. After transference, the membrane was incubated with 5% nonfat milk in TBST (10 mM Tris (pH 8.0), 150 mM NaCl, 0.5% Tween 20) for 2 h at room temperature, then washed three times with TBST and incubated with the obtained rabbit IgG antibodies (1:50) for 1 h, rewashed three times with TBST, and finally incubated with the second antibody (goat anti-rabbit IgG coupled to alkaline-phosphatase at 1:2000). The membrane was washed with TBST three times and developed with 3,3 ,5,5 -tetramethylbenzidine (TMB) ready-to-use solution (Invitrogen Antibodies, Thermo Fisher Scientific, Asheville, NC, USA) according to the manufacturer's protocols.

Animal Immunizations
Rabbits were hyperimmunized subcutaneously with 7.3 mg of HisrCrotoxin B to raise serum antibodies (anti-HisrCrotoxin B). The immunization protocol started with 0.02 mg total protein in Complete Freud's Adjuvant (CFA). Increasing doses up to 0.64 mg, alternating between incomplete Freud's (IFA) and aluminum hydroxide (AH), were applied for 5 months, besides 4 boost applications without adjuvant injected every month along with the immunization. The rabbit serum antibodies were purified from plasma by acid precipitation using 5% caprylic acid.

Enzyme-Linked Immunosorbent Assay (ELISA)
Solid-phase-adsorbed MaxiSorp plates (NUNC™, Thermo scientific, Waltham, MA, USA) were prepared by treating wells with a 100 µL solution of 5 µg/mL of each protein (HisrCrotoxin B, native Crotoxin B, and venoms) in 100 mM sodium carbonate buffer (pH 9.6). After overnight incubation at 4 • C, the wells were aspirated and washed three times with 200 µL of a washing buffer (50 mM Tris-HCl (pH 8), containing 0.5 mg/mL of Tween 20 and 150 mM NaCl). The wells were then filled with 200 µL of blocking buffer (5 mg/mL gelatin and 2 mg/mL Tween 20 in 50 mM Tris-HCl buffer at pH 8). After 2 h incubation at 37 • C, the wells were washed, as described above, and filled with 100 µL of serially diluted rabbit IgG anti-HisrCrotoxin B in incubation buffer (50 mM Tris-HCl buffer at pH 8 containing 1 mg/mL gelatin, 0.5 mg/mL Tween 20, and 0.5 M NaCl). The starting dilution was 1:30, from an IgG concentration of 4 mg/mL, and the incubation time was 1 h at 37 • C. After washing, the bound rabbit IgGs were allowed to react with 100 µL per well of 0.1 mg/mL of anti-rabbit IgGs labeled with horseradish peroxidase (Roche, Basel, Switzerland) in the incubation buffer. After 1 h at 37 • C, wells were washed and filled with 100 µL of 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) solution (Roche, Gaithersburg MD, USA) as the substrate for peroxidase. The color development reaction was stopped by adding 25 µL of 20% sodium dodecyl sulfate (SDS), and the plate was read at 405 nm in a Microplate Reader (Tecan Sunrise IVD version, Chapel Hill, NC, USA). Data were analyzed by nonlinear regression using the sigmoidal dose-response equation of the Prism program (Graph Pad Prism v. 6.0c, San Diego, CA, USA). Conventional titers were calculated from the midpoint of the curve and correspond to the IgG dilution for half of the maximal binding, and it was considered the half-maximal inhibitory concentration (IC 50 ).

Phospholipase Activity
The hydrolysis of egg yolk at 10% determined the phospholipase activity of the purified enzyme in a buffer (0.1 M NaCl, 0.01 M CaCl 2 , 0.5% Triton X-100). The buffer's pH was set to 8.05, and the sample was added. We measured the time when the pH value dropped to 7.99, and then 0.05 M NaOH was used to up the pH value again. We continued measuring the time when the pH value dropped again in a total of 5 repetitions. Slight N 2 bubbling was applied during the assay to avoid the O 2 intervention in the reaction. The activity was determined as the µmol of NaOH consumed per minute per mg of protein.

Neutralization Activity
The protocol used for assaying the activity of HisrCrotoxin B in vivo, using a mouse model, was followed according to our Institute Committee of Animal Welfare guidelines, keeping the number of animals to minimum required to validate the experiments. Male mice (CD-1, 18-20 g body weight) were tested by intravenous injection.

Statistics
Results were expressed as mean and standard deviation or as mean with 95% confidence intervals. The software Prism 4.0 (Graph Pad Inc., San Diego, CA, USA) was used for all statistical methods.